Differential <i>N</i>‐ethylmaleimide inhibition of two enzymes of the DNA α‐polymerase‐fraction from calf thymus

Wickremasinghe, RG; Hesslewood, Ian P.; Holmes, Andrew M.; Johnston, Irving R.

doi:10.1016/0014-5793(77)80291-3

Cited by 9 publications

(5 citation statements)

References 14 publications

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“…Incubation of the 9S polymerase with 0.01 mM NEM for 10 min at 37 °C destroys half of the activity. The NEM sensitivity of the 9S enzyme is considerably greater than that of the 8S enzyme reported by Wickremasinghe et al (1977); it is comparable, however, to that of the Drosophila enzyme (Banks et al, 1979).…”

Section: Resultscontrasting

confidence: 55%

Purification of a 9S DNA polymerase .alpha. species from calf thymus

Grosse¹,

Krauss²

1981

Biochemistry

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a DNA polymerase alpha species from calf thymus has been purified 12 000-fold to near homogeneity. The enzyme sediments under high salt conditions in the preparative ultracentrifuge as a homogeneous band at 9S. The specific activity is 50 000-70 000 units/mg of protein. Polypeptides of 148 000, 59 000, and 48 000 daltons are detectable. The molecular weight as estimated from gradient gel electrophoresis is about 500 000. The 9S DNA polymerase is free from terminal nucleotidyl transferase activity and does not exhibit endonuclease or exonuclease activity. It is inhibited by low concentrations of salt, aphidicolin, and N-ethylmaleimide.

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Section: Resultscontrasting

confidence: 55%

Purification of a 9S DNA polymerase .alpha. species from calf thymus

Grosse¹,

Krauss²

1981

Biochemistry

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“…Although it is premature to determine a correspondence between the partially purified placental a species and those obtained in other systems by procedures which are not identical with those described here, our extraction procedures when applied to IMR-90-cultured human fibroblasts yield two fractions which appear to be analogous to a¡ and a2. Multiple -polymerases have also been observed in murine, avian, and bovine tissues (Hackmann & Lezius, 1975;Fry & Weisman-Shomer, 1976;Matsukage et al, 1976;Pedrali-Noy & Weissbach, 1977;Wickremasinghe et al, 1977) and recently in a transformed human cell line from which one species has been purified to homogeneity (Fisher & Korn, 1977). The heterogeneity could be physiologically significant because an increase in the activity of -polymerase correlates with increased cellular and viral DNA synthesis (Pritchard et al, 1978;Edenberg et al, 1978;Chiu & Baril, 1975;Hiibscher et al, 1977).…”

Section: Discussionmentioning

confidence: 99%

Fidelity of fractionated deoxyribonucleic acid polymerases from human placenta

Krauss¹,

Linn²

1980

Biochemistry

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“…A series of experiments was undertaken to determine whether the presence of singlestranded DNA or single-stranded DNA oligomers was capable of protecting the DNA-binding activity of ICP8 from the action of NEM. This seemed to be a reasonable possibility, since Wickremasinghe et al (36) found that the presence of activated DNA resulted in the protection of the activity of mammalian DNA polymerase from inactivation by NEM. In one set of experiments, ICP8 at a concentration of 128 jig/ml was incubated at room temperature in the presence of 10 mM NEM.…”

Section: Resultsmentioning

confidence: 99%

N-ethylmaleimide inhibition of the DNA-binding activity of the herpes simplex virus type 1 major DNA-binding protein

Ruyechan

1988

J Virol

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The major herpes simplex virus DNA-binding protein, designated ICP8, binds tightly to single-stranded DNA and is required for replication of viral DNA. The sensitivity of the DNA-binding activity of ICP8 to the action of the sulfhydryl reagent N-ethylmaleimide has been examined by using nitrocellulose filter-binding and agarose gel electrophoresis assays. Incubation of ICP8 with N-ethylmaleimide results in a rapid loss of DNA-binding activity. Preincubation of ICP8 with single-stranded DNA markedly inhibits this loss of binding activity. These results imply that a free sulfiydryl group is involved in the interaction of ICP8 with single-stranded DNA and that this sulfhydryl group becomes less accessible to the environment upon binding. Agarose gel electrophoretic analysis of the binding interaction in the presence and absence of N-ethylmaleimide indicates that the cooperative binding exhibited by ICP8 is lost upon treatment with this reagent but that some residual noncooperative binding may remain. This last result was confirmed by equilibrium dialysis

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Differential N‐ethylmaleimide inhibition of two enzymes of the DNA α‐polymerase‐fraction from calf thymus

Cited by 9 publications

References 14 publications

Purification of a 9S DNA polymerase .alpha. species from calf thymus

Purification of a 9S DNA polymerase .alpha. species from calf thymus

Fidelity of fractionated deoxyribonucleic acid polymerases from human placenta

N-ethylmaleimide inhibition of the DNA-binding activity of the herpes simplex virus type 1 major DNA-binding protein

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